High Speed Confocal Photomanipulation Microscopy for use in multi-user facility Project Summary Protein function depends on the availability of a protein to interact with substrates or partner proteins in the cellular environment. By labeling proteins with green fluorescent protein and applying laser scanning confocal microscopy, it has become possible to directly quantitate protein availability in live cells. Selective photobleaching or photoactivation (photomanipulation) of a population of fluorescently labeled proteins permits investigators to measure a protein's mobility, molecular size, percent of mobile proteins, and follow a photoactivated protein's fate. While fluorescence imaging and photomanipulation have provided many insights into protein dynamics, a serious deficit in this methodology has been the relatively slow rates of data acquisition (tenths of seconds per image) and the delay between the end of a photomanipulation event and the collection of the first postmanipulation images. This is especially problematic for cytoplasmic or lumenal proteins that can move several microns in the space of milliseconds. A new confocal photomanipulation microscope from Zeiss, the Duo, has broken this temporal barrier with remarkable image quality, quantum efficiency, and speed. The Zeiss Duo system proposed here will be part of a multi-user facility and cannot be used exclusively by one laboratory. The purpose of this application is to provide technology to the AECOM community that is not currently available. None of the equipment currently on campus is suited to the simultaneous high speed confocal imaging coupled with rapid region of interest photobleaching or photoactivation of live material for protein mobility measurements. The common scientific need of the major users in this application is the ability to obtain high spatial and temporal resolution images from live cells for quantitative protein mobility measurements. As discussed in the budget justification, the system we request will solve the problems of speed, sensitivity, and quantitation for an expert user group. Relevance: Mis-regulation of cellular proteins occurs in diseases ranging from cancer to Alzheimer's disease. To define the normal and disease properties of cellular proteins, it is critical to identify the environments, binding partners, and dynamics of cellular proteins. The high speed microscope described in this proposal will fulfill this need for our research groups. [unreadable] [unreadable] [unreadable]